No description available.

Com o intuito de investigar se a hipóxia representa um dos desencadeadores da regressão luteínica em cadelas não prenhes, o presente estudo foi delineado para analisar a expressão do fator transcricional indutível por hipóxia HIF1A e a de seus genes-alvo relacionados à angiogênese, como o fator de crescimento endotelial vascular (VEGFA) e à captação de glicose, como as proteínas transportadoras facilitadoras GLUT1/SLC2A1 e GLUT4/SLC2A4 no corpo lúteo canino ao longo do diestro (dias 10 a 70 após a ovulação). Para tal, utilizou-se imuno-histoquímica e western blotting para localizar e quantificar as proteínas do HIF1A, GLUT1 e GLUT4 e PCR em tempo real para quantificar a expressão do RNAm de HIF1A, SLC2A1, SLC2A4, VEGFA, FLT1 e KDR. Além disso, células luteínicas nas fases inicial (dia 10), média (dia 30) e final (dia 60) foram submetidas ao tratamento com cloreto de cobalto (CoCl2) a 500 µM para avaliar os efeitos da hipóxia sobre a expressão gênica dos fatores acima citados, bem como sobre a produção de progesterona e 17β-estradiol. Nossos resultados demonstraram que o HIF1A é expresso pelo corpo lúteo canino de maneira tempo-dependente ao longo do diestro, e que a expressão de seu RNAm está diretamente correlacionada a expressão gênica de SLC2A1, SLC2A4, VEGFA, FLT1 e KDR e com as concentrações de progesterona periférica. No cultivo primário de células luteínicas, a hipóxia induzida pelo CoCl2 diminuiu a produção de progesterona e de 17β-estradiol e estimulou significativamente a expressão de HIF1A, SLC2A1, SLC2A4 e VEGFA. Esses resultados sugerem que o HIF1A constitui um dos fatores regulatórios da função do corpo lúteo canino participando da modulação de processos como esteroidogêne, angiogênese e da captação de glicose, atuando como fator luteolítico. / This study was designed to investigate if hypoxia is one of the triggers of luteal regression in non-pregnant bitches. For that, we analyzed the hypoxia- inducible factor (HIF1A) expression as well as the expression of its target genes related to angiogenesis (vascular endothelial growth factor VEGFA) and to glucose uptake (glucose transporters GLUT/SLC2A 1 and 4) in canine corpus luteum throughout diestrus (days 10 to 70 after ovulation). We used immunohistochemistry and western blotting to localize and quantify the protein expression of HIF1A, GLUT1 and GLUT4, respectively, and real time PCR to analyze HIF1A, SLC2A1, SLC2A4, VEGFA, FLT1 and KDR gene expression. Moreover, luteal cells from early (day 10), mid (day 30) and late luteal phase (day 60) were submitted to 500 µM cobalt chloride (CoCl2) treatment to verify hypoxia effects on gene expression of the above cited genes and on progesterone and 17β-estradiol production. Our results showed that luteal cells expressed HIF1A in a time-dependent manner over diestrus and that its expression was directly correlated to both SLC2A1, SLC2A4, VEGFA, FLT1 and KDR gene expression and progesterone production. The protein expression of the studied genes also changed over diestrus and was correlated with the respective gene expression. In primary luteal cells culture, cobalt chloride-induced hypoxia downregulated progesterone and 17β-estradiol production, but upregulated HIF1A, SLC2A1, SLC2A4 and VEGFA gene expression. These findings suggest that HIF1A is one of the factors regulating canine luteal function by modulating important process as steroidogenesis, angiogenesis and glucose uptake, acting as a pro-survival factor.

Com o intuito de investigar se a hipóxia representa um dos desencadeadores da regressão luteínica em cadelas não prenhes, o presente estudo foi delineado para analisar a expressão do fator transcricional indutível por hipóxia HIF1A e a de seus genes-alvo relacionados à angiogênese, como o fator de crescimento endotelial vascular (VEGFA) e à captação de glicose, como as proteínas transportadoras facilitadoras GLUT1/SLC2A1 e GLUT4/SLC2A4 no corpo lúteo canino ao longo do diestro (dias 10 a 70 após a ovulação). Para tal, utilizou-se imuno-histoquímica e western blotting para localizar e quantificar as proteínas do HIF1A, GLUT1 e GLUT4 e PCR em tempo real para quantificar a expressão do RNAm de HIF1A, SLC2A1, SLC2A4, VEGFA, FLT1 e KDR. Além disso, células luteínicas nas fases inicial (dia 10), média (dia 30) e final (dia 60) foram submetidas ao tratamento com cloreto de cobalto (CoCl2) a 500 µM para avaliar os efeitos da hipóxia sobre a expressão gênica dos fatores acima citados, bem como sobre a produção de progesterona e 17β-estradiol. Nossos resultados demonstraram que o HIF1A é expresso pelo corpo lúteo canino de maneira tempo-dependente ao longo do diestro, e que a expressão de seu RNAm está diretamente correlacionada a expressão gênica de SLC2A1, SLC2A4, VEGFA, FLT1 e KDR e com as concentrações de progesterona periférica. No cultivo primário de células luteínicas, a hipóxia induzida pelo CoCl2 diminuiu a produção de progesterona e de 17β-estradiol e estimulou significativamente a expressão de HIF1A, SLC2A1, SLC2A4 e VEGFA. Esses resultados sugerem que o HIF1A constitui um dos fatores regulatórios da função do corpo lúteo canino participando da modulação de processos como esteroidogêne, angiogênese e da captação de glicose, atuando como fator luteolítico. / This study was designed to investigate if hypoxia is one of the triggers of luteal regression in non-pregnant bitches. For that, we analyzed the hypoxia- inducible factor (HIF1A) expression as well as the expression of its target genes related to angiogenesis (vascular endothelial growth factor VEGFA) and to glucose uptake (glucose transporters GLUT/SLC2A 1 and 4) in canine corpus luteum throughout diestrus (days 10 to 70 after ovulation). We used immunohistochemistry and western blotting to localize and quantify the protein expression of HIF1A, GLUT1 and GLUT4, respectively, and real time PCR to analyze HIF1A, SLC2A1, SLC2A4, VEGFA, FLT1 and KDR gene expression. Moreover, luteal cells from early (day 10), mid (day 30) and late luteal phase (day 60) were submitted to 500 µM cobalt chloride (CoCl2) treatment to verify hypoxia effects on gene expression of the above cited genes and on progesterone and 17β-estradiol production. Our results showed that luteal cells expressed HIF1A in a time-dependent manner over diestrus and that its expression was directly correlated to both SLC2A1, SLC2A4, VEGFA, FLT1 and KDR gene expression and progesterone production. The protein expression of the studied genes also changed over diestrus and was correlated with the respective gene expression. In primary luteal cells culture, cobalt chloride-induced hypoxia downregulated progesterone and 17β-estradiol production, but upregulated HIF1A, SLC2A1, SLC2A4 and VEGFA gene expression. These findings suggest that HIF1A is one of the factors regulating canine luteal function by modulating important process as steroidogenesis, angiogenesis and glucose uptake, acting as a pro-survival factor.

Glucose is a monosaccharide and fuel for body, it cannot pass through membrane by simple diffusion so, integral transmembrane proteins named glucose transporters (Gluts) are involved in the regulation of the movement of glucose between the extracellular and intracellular spaces within the body. GLUT1 and GLUT3 have previously been shown by cold detergent extraction methods to reside in distinct plasma membrane domains in non-polarized mammalian cells, with GLUT1, but not GLUT3, residing  in detergent-resistant membrane (DRM) domains. To confirm this observation under less invasive conditions, molecular fusion tags are inserted in the first external loop in Glut1 with biotin ligase acceptor peptide (BLAP) between Ser-55 and Ile-56 and in Glut3 with Acyl carrier peptide (ACP) in between Val-57 and Leu-58 respectively. These Glut fusion proteins will be used in order to confirm these observations by fluorescence recovery after photobleaching (FRAP) and single molecule fluorescence microscopy in live cells. hGLUT1-EGFP, hGLUT1 (AgeI)-EGFP recombinants were constructed and transfected human embryonic kidney cells (HEK-293) quantum dot images supports the fact that EGFP transfected cells uniformly and is distributed in the cell cytoplasm, hGLUT1-EGFP transfected cells and is localized to the cell membrane and hGLUT1 (AgeI)-EGFP transfected cells and located to the plasma membrane with high intensity.

<p>Glucose is a monosaccharide and fuel for body, it cannot pass through membrane by simple diffusion so, integral transmembrane proteins named glucose transporters (Gluts) are involved in the regulation of the movement of glucose between the extracellular and intracellular spaces within the body. GLUT1 and GLUT3 have previously been shown by cold detergent extraction methods to reside in distinct plasma membrane domains in non-polarized mammalian cells, with GLUT1, but not GLUT3, residing  in detergent-resistant membrane (DRM) domains. To confirm this observation under less invasive conditions, molecular fusion tags are inserted in the first external loop in Glut1 with biotin ligase acceptor peptide (BLAP) between Ser-55 and Ile-56 and in Glut3 with Acyl carrier peptide (ACP) in between Val-57 and Leu-58 respectively. These Glut fusion proteins will be used in order to confirm these observations by fluorescence recovery after photobleaching (FRAP) and single molecule fluorescence microscopy in live cells. hGLUT1-EGFP, hGLUT1 (<em>AgeI</em>)-EGFP recombinants were constructed and transfected human embryonic kidney cells (HEK-293) quantum dot images supports the fact that EGFP transfected cells uniformly and is distributed in the cell cytoplasm, hGLUT1-EGFP transfected cells and is localized to the cell membrane and hGLUT1 (<em>AgeI</em>)-EGFP transfected cells and located to the plasma membrane with high intensity.</p>

No description available.